LARS Antibody, HRP conjugated

What is LARS Antibody, HRP conjugated and why is it significant in research?

LARS Antibody, HRP conjugated is a polyclonal antibody that specifically targets Leucine--tRNA ligase (LARS), a cytoplasmic protein involved in protein synthesis. The antibody is raised in rabbits using a recombinant human LARS protein fragment (amino acids 614-892) as the immunogen . The antibody has been conjugated with Horseradish Peroxidase (HRP), a 44 kDa glycoprotein containing 6 lysine residues that facilitates visualization through chromogenic reactions .

The significance of HRP conjugation lies in its ability to enable direct detection without requiring secondary antibodies, thereby avoiding potential cross-species reactivity issues and eliminating additional wash and separation steps in experimental protocols . This direct detection capability is particularly valuable in time-sensitive applications and complex experimental designs where minimizing variables is critical.

What cellular processes does LARS participate in, and how does this inform antibody applications?

LARS (Leucine--tRNA ligase, cytoplasmic) is fundamentally involved in protein synthesis as it catalyzes the attachment of leucine to its corresponding tRNA molecules (EC 6.1.1.4) . This aminoacyl-tRNA synthetase plays a crucial role in translational fidelity. Recent research has expanded our understanding of LARS beyond its canonical role in translation, with emerging evidence suggesting involvement in cellular signaling pathways and potential regulatory functions.

The LARS antibody can be applied in various research contexts, including:

• Investigation of protein synthesis machinery

• Studies of translational control mechanisms

• Research on amino acid sensing and nutrient signaling

• Analysis of potential non-canonical functions of aminoacyl-tRNA synthetases

The HRP conjugation makes this antibody particularly suitable for ELISA applications, as indicated in product specifications .

How does the reactivity profile of LARS Antibody, HRP conjugated influence experimental design?

The LARS Antibody, HRP conjugated has been specifically validated for human reactivity . This species-specific reactivity profile is a critical consideration when designing experiments, as it determines which experimental models and systems are suitable. Researchers working with human cell lines, tissues, or clinical samples can confidently employ this antibody, knowing it has been tested for human LARS detection.

When designing experiments with this antibody, researchers should:

• Select appropriate human-derived experimental systems

• Include proper positive controls (human samples expressing LARS)

• Consider potential cross-reactivity limitations if working with non-human models

• Validate the antibody's performance in their specific experimental context before proceeding with larger studies

What buffer conditions optimize the performance of LARS Antibody, HRP conjugated?

The performance of LARS Antibody, HRP conjugated is significantly influenced by buffer composition. The antibody is provided in a storage buffer containing 0.03% Proclin 300 as a preservative, 50% Glycerol, and 0.01M PBS at pH 7.4 . When designing experimental buffers for optimal antibody performance, researchers should consider the following parameters based on general HRP antibody guidance:

Additionally, certain components should be strictly avoided in working solutions, including thiomersal/thimerosal, merthioloate, sodium azide, glycine, proclin, and nucleophilic components such as primary amines and thiols, as these can significantly compromise HRP activity .

What are the critical storage parameters for maintaining LARS Antibody, HRP conjugated activity?

Proper storage is essential for maintaining the activity and specificity of LARS Antibody, HRP conjugated. According to product specifications, the antibody should be stored at -20°C or -80°C upon receipt . Importantly, repeated freeze-thaw cycles should be avoided as they can lead to protein denaturation and loss of activity.

For optimal preservation of HRP-conjugated antibodies:

• Store the stock antibody at the recommended temperature (-20°C or -80°C)

• Aliquot the antibody into single-use volumes before freezing to avoid repeated freeze-thaw cycles

• When thawing, allow the antibody to equilibrate to room temperature gradually

• For working dilutions, use freshly prepared buffers free from contaminants that could affect HRP activity

• Consider using stabilizers such as LifeXtendTM for diluted working solutions to protect against performance loss

It's important to note that even with optimal storage, the performance of HRP conjugates naturally diminishes over time, with the degradation rate accelerating at higher temperatures and in more dilute solutions .

What detection systems provide optimal visualization of LARS Antibody, HRP conjugated?

Horseradish peroxidase conjugated to the LARS antibody offers versatile detection options through various chromogenic and chemiluminescent substrates. The choice of detection system depends on the specific research requirements, including sensitivity needs, equipment availability, and whether quantitative or qualitative data is desired.

Common detection substrates for HRP include:

• Diaminobenzidine (DAB): When exposed to hydrogen peroxide (H₂O₂), DAB is converted into a water-insoluble brown pigment, making it ideal for immunohistochemistry applications .

• ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)): Produces a soluble green product, suitable for ELISA and other solution-based assays.

• TMB (3,3',5,5'-tetramethylbenzidine): Generates a blue product that turns yellow when stopped with acid, providing excellent sensitivity for ELISA.

• TMBUS: Another substrate option for measuring horseradish peroxidase activity .

• Enhanced chemiluminescent (ECL) substrates: For applications requiring higher sensitivity or when using imaging systems rather than spectrophotometric detection.

For quantitative applications like ELISA, which is the validated application for this LARS antibody , TMB often provides an excellent balance of sensitivity and ease of use with straightforward spectrophotometric readout.

How can researchers validate the specificity of LARS Antibody, HRP conjugated?

Validating antibody specificity is a critical step in ensuring experimental rigor. For LARS Antibody, HRP conjugated, researchers should implement a multi-faceted validation approach:

• Positive control: Use cells or tissues known to express LARS, such as human cell lines with documented LARS expression.

• Knockout/knockdown verification: Compare signals between wild-type samples and those where LARS has been knocked out or knocked down via CRISPR or siRNA.

• Peptide competition assay: Pre-incubate the antibody with the immunogen peptide (recombinant Human Leucine--tRNA ligase, cytoplasmic protein, amino acids 614-892) before application to samples. A specific antibody will show reduced or eliminated signal.

• Molecular weight verification: Confirm that the detected protein migrates at the expected molecular weight for LARS in western blot applications.

• Comparison with alternative antibodies: If possible, compare results with other validated LARS antibodies targeting different epitopes.

What are common sources of background when using LARS Antibody, HRP conjugated, and how can they be mitigated?

High background is a common challenge when working with HRP-conjugated antibodies like the LARS Antibody. Several sources and corresponding mitigation strategies include:

• Non-specific binding:

  • Cause: Insufficient blocking or antibody concentration too high

  • Solution: Optimize blocking agents (BSA, normal serum, or commercial blockers) and titrate antibody dilutions carefully

• Endogenous peroxidase activity:

  • Cause: Sample contains endogenous peroxidases that react with the substrate

  • Solution: Include a peroxidase quenching step (e.g., 0.3% H₂O₂ in methanol) before antibody application

• Cross-reactivity:

  • Cause: Antibody binding to proteins with similar epitopes

  • Solution: Use more stringent washing conditions and validate specificity as outlined in section 3.1

• Suboptimal buffer composition:

  • Cause: Buffer components interfering with antibody-antigen interaction

  • Solution: Ensure working buffers adhere to the recommendations in section 2.1, avoiding components that interfere with HRP activity

• Sample overfixation:

  • Cause: Excessive fixation masking epitopes or causing non-specific binding

  • Solution: Optimize fixation protocols and consider implementing antigen retrieval techniques

Implementing a systematic approach to troubleshooting, where one parameter is modified at a time, will help identify and resolve specific sources of background.

How can the signal-to-noise ratio be optimized in ELISA using LARS Antibody, HRP conjugated?

Optimizing signal-to-noise ratio is crucial for reliable results in ELISA using LARS Antibody, HRP conjugated. A methodical approach includes:

• Antibody titration: Perform a checkerboard titration to identify the optimal antibody concentration that yields maximum specific signal with minimal background.

• Buffer optimization:

  • Use recommended buffer conditions (pH 6.5-8.5)

  • Keep BSA and gelatin levels below 0.1%

  • Ensure Tris concentration remains below 50mM

• Blocking protocol refinement:

  • Test different blocking agents (BSA, milk protein, commercial blockers)

  • Optimize blocking time and temperature

  • Consider specialized blocking agents for problematic samples

• Washing optimization:

  • Increase number of washes

  • Add detergents (e.g., 0.05% Tween-20) to wash buffers

  • Use multi-channel pipettes or automated washers for consistent washing

• Substrate selection and development:

  • Choose appropriate substrate based on required sensitivity

  • Optimize substrate incubation time

  • Consider kinetic readings to capture optimal signal window

• Sample preparation:

  • Ensure samples are properly prepared to maximize target accessibility

  • Remove potential interfering components through additional purification steps if necessary

Implementing these optimizations systematically will help achieve reliable and reproducible results with high signal-to-noise ratios.

How can LARS Antibody, HRP conjugated be integrated into multiplex detection systems?

Integrating LARS Antibody, HRP conjugated into multiplex detection systems requires careful consideration of detection compatibility and potential cross-reactivity. Although traditional HRP-based detection typically generates a single-channel readout, several strategies can enable multiplexing:

• Sequential multiplexing approach:

  • Perform complete LARS detection with HRP conjugate first

  • Document results thoroughly

  • Strip the antibody using appropriate stripping buffer

  • Proceed with detection of subsequent targets using different visualization systems

• Spatial segregation multiplexing:

  • Apply LARS Antibody, HRP conjugated to discrete sections or wells

  • Utilize other detection methods in separate compartments

  • Analyze results comparatively through computational integration

• Combinatorial detection:

  • Use HRP detection for LARS in combination with fluorescent or other enzyme-based systems for other targets

  • Select visualization substrates that produce spectrally distinct readouts

  • Employ appropriate imaging systems capable of distinguishing multiple signals

• Advanced molecular barcoding:

  • Combine HRP-based detection with molecular barcoding techniques

  • Link results through computational algorithms and machine learning approaches

  • Integrate spatial and molecular information for comprehensive analysis

When implementing these approaches, researchers must validate each detection system independently before combining them, ensuring no interference between detection modalities.

What methodological considerations apply when using LARS Antibody, HRP conjugated for protein-protein interaction studies?

Investigating protein-protein interactions involving LARS using HRP-conjugated antibodies requires specialized methodological considerations:

• Co-immunoprecipitation optimization:

  • Use mild lysis buffers to preserve protein-protein interactions

  • Consider cross-linking approaches to stabilize transient interactions

  • Implement controls to distinguish specific from non-specific interactions

  • Optimize wash stringency to balance removal of non-specific binding with preservation of genuine interactions

• Proximity ligation assays:

  • Combine LARS Antibody, HRP conjugated with antibodies against potential interaction partners

  • Utilize specialized detection systems that generate signal only when proteins are in close proximity

  • Implement appropriate negative controls (proteins known not to interact with LARS)

• HRP activity considerations:

  • Ensure HRP conjugation doesn't interfere with recognition of interaction interfaces

  • Consider using alternative detection methods if steric hindrance is suspected

  • Validate findings using complementary approaches (e.g., pull-down assays with recombinant proteins)

• Binding kinetics analysis:

  • Develop quantitative assays to measure association and dissociation rates

  • Consider temperature and buffer condition effects on interaction stability

  • Implement dose-response studies to characterize interaction dynamics

These methodologies should be adapted to the specific research question, with careful consideration of control experiments to validate findings.

How does HRP conjugation affect the binding kinetics of LARS Antibody compared to other detection methods?

The conjugation of HRP to LARS Antibody can potentially influence its binding characteristics compared to unconjugated or differently labeled antibodies. Understanding these effects is crucial for accurate interpretation of experimental results:

Researchers should conduct comparative studies using the same antibody with different detection methods when binding kinetics are crucial to experimental interpretation.

What advanced quantitative approaches can improve analysis of LARS expression using HRP-conjugated antibodies?

Advanced quantitative analysis of LARS expression using HRP-conjugated antibodies can benefit from sophisticated methodological approaches:

• Digital image analysis for immunohistochemistry:

  • Implement machine learning algorithms for automated quantification

  • Utilize color deconvolution to separate DAB signal from counterstains

  • Apply threshold-independent quantification methods

  • Correlate staining intensity with calibrated standards

• Enzyme kinetics-based quantification:

  • Measure initial reaction velocities rather than endpoint measurements

  • Develop standard curves using recombinant LARS protein

  • Implement Michaelis-Menten kinetic analysis for substrate conversion

  • Account for potential enzyme inactivation during extended incubations

• Multiplexed internal standards:

  • Include calibrated reference proteins for normalization

  • Develop ratio-metric quantification approaches

  • Implement spike-in controls with known concentrations

• Advanced ELISA techniques:

  • Adapt traditional ELISA to include kinetic measurements

  • Implement digital ELISA approaches for single-molecule detection

  • Develop competition assays for improved quantification

• Computational integration:

  • Combine data from multiple experimental approaches

  • Implement Bayesian statistical frameworks for improved estimation

  • Develop computational models integrating expression data with functional readouts

These advanced approaches can significantly enhance the quantitative rigor of experiments using LARS Antibody, HRP conjugated, enabling more precise characterization of LARS expression levels across various experimental conditions.

How does LARS Antibody, HRP conjugated compare with other available LARS antibodies?

The research antibody marketplace offers various LARS antibodies with different configurations and applications. LARS Antibody, HRP conjugated (CSB-PA873736LB01HU) should be evaluated in context of alternatives:

When selecting between these options, researchers should consider:

• The specific application requirements (technique, sensitivity needs)

• The experimental model system (human vs. other species)

• The availability of detection systems and preference for direct vs. indirect detection

• The need for specific epitope targeting (N-terminal vs. C-terminal)

For applications focused specifically on ELISA with human samples, the HRP-conjugated variant offers streamlined protocols, while unconjugated alternatives provide greater flexibility across different applications.

What validation methods are essential before implementing LARS Antibody, HRP conjugated in novel research contexts?

Before implementing LARS Antibody, HRP conjugated in novel research contexts, comprehensive validation is essential to ensure reliability of results:

• Epitope-specific validation:

  • Verify recognition of the intended epitope region (amino acids 614-892 of human LARS)

  • Perform peptide competition assays using the immunogen

  • Compare results with antibodies targeting different LARS epitopes

• Application-specific verification:

  • Though validated for ELISA , test performance in the specific ELISA format being used

  • Establish optimal working dilutions for each specific application

  • Determine sensitivity limits through serial dilution of positive control samples

• Specificity confirmation:

  • Test with samples containing varying LARS expression levels

  • Include negative controls (LARS-knockout or -knockdown samples)

  • Assess potential cross-reactivity with related proteins (other aminoacyl-tRNA synthetases)

• Reproducibility assessment:

  • Conduct inter-assay and intra-assay variation analysis

  • Test across different sample preparation methods

  • Evaluate lot-to-lot consistency if using the antibody long-term

• Context-dependent optimization:

  • Adjust protocols based on specific sample types (cell lysates, tissue extracts, etc.)

  • Optimize buffer conditions for specific experimental contexts

  • Determine if antigen retrieval or other specialized treatments are needed

These validation steps ensure that findings generated using LARS Antibody, HRP conjugated are reliable and reproducible, particularly important when extending its use beyond manufacturer-validated applications.

What emerging research areas could benefit from LARS Antibody, HRP conjugated investigations?

Several cutting-edge research areas could benefit from investigations using LARS Antibody, HRP conjugated:

• Non-canonical functions of aminoacyl-tRNA synthetases:

  • Investigating LARS involvement in signaling pathways beyond protein synthesis

  • Exploring potential regulatory roles in nutrient sensing

  • Examining interactions with components outside the translation machinery

• Cancer metabolism and protein synthesis regulation:

  • Characterizing LARS expression changes across cancer types

  • Exploring connections between LARS activity and cancer cell proliferation

  • Investigating LARS as a potential therapeutic target or biomarker

• Cellular stress response mechanisms:

  • Analyzing LARS localization and expression under various stress conditions

  • Investigating potential involvement in integrated stress response pathways

  • Examining connections to mTOR signaling and autophagy regulation

• Neurodegenerative disease mechanisms:

  • Exploring LARS expression in neurological disorders

  • Investigating potential connections to protein misfolding diseases

  • Examining roles in maintaining proteostasis in neuronal cells

• Epigenetic regulation:

  • As indicated by the Research Area classification (Epigenetics and Nuclear Signaling)

  • Investigating potential nuclear functions of LARS

  • Exploring connections between amino acid metabolism and epigenetic modifications

These emerging research directions highlight the importance of reliable tools like LARS Antibody, HRP conjugated for exploring the diverse biological roles of aminoacyl-tRNA synthetases beyond their canonical functions in translation.

How can researchers optimize LARS Antibody, HRP conjugated protocols for automated high-throughput screening?

Adapting LARS Antibody, HRP conjugated for automated high-throughput screening requires systematic optimization:

• Miniaturization strategies:

  • Scale protocols to microplate formats (96, 384, or 1536-well)

  • Optimize reagent volumes to maintain signal while reducing consumption

  • Validate signal consistency across well positions to identify edge effects

• Automation compatibility:

  • Ensure buffers are compatible with liquid handling systems

  • Minimize protocol steps requiring manual intervention

  • Develop robust quality control metrics for automated processes

• Assay stability considerations:

  • Test working dilution stability at room temperature over time

  • Implement stabilizers (such as LifeXtendTM) for maintaining HRP activity

  • Develop standard operating procedures for reagent preparation and storage

• Detection optimization:

  • Select substrates compatible with automated plate readers

  • Optimize incubation times for the dynamic range of detection instruments

  • Implement internal controls for normalization across plates

• Data analysis pipelines:

  • Develop automated data processing workflows

  • Implement quality control metrics for assay performance

  • Design statistical approaches appropriate for high-throughput data

By systematically addressing these aspects, researchers can successfully transition protocols using LARS Antibody, HRP conjugated from manual to automated high-throughput formats while maintaining assay performance and reliability.